Team:Caltech/Protocols/Folate assay

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Folate Microbiological Assay Protocol

Purpose

To quantify and measure levels of folate production in the cell lysate and supernatant of 'E.coli' transformed with high copy folate biosynthesis genes 'folB,' 'folKE,' 'folBKE'.

Materials

• Folic Acid Assay media
• Lactobacilli Broth AOAC
• Folic Acid (for standard curve)
• 0.1M sodium acetate buffer (pH4.8) -1% ascorbic acid
  • Ascorbic acid (Sigma, A4544-25G)
• 0.1M 2-mercaptoethanol-0.5% sodium ascorbate (We used the above 0.1M sodium acetate - 1% ascorbic acid buffer in place of the sodium ascorbate)(for deconjugation mix)
• Human plasma (Sigma, P9523-1ML)(for deconjugation mix)
• Lactobacillus rhamnosus (ATCC 7469)

Deconjugation Mixture Sybesma

1. Dilute 1 g of human plasma in 5ml of 0.1M 2-mercaptoethanol-0.5% sodium ascorbate

2. Clear from precipitates by centrifugation (10,000xg,2 min)

3. Add 2.5% (vol/vol) concentration of the clarified human plasma solution to the folate samples.

Sample Preparation

Standard Curve

Notes

• Previously, we had been using this protocol with the other folate-dependent strain Entercoccus hirae ATCC 8043, and were able to generate a linear standard curve from 0.1-1ng. However, this strain did not grow in our samples, and so we switched to L. rhamnosus.
• This is why we used Lactobacilli Broth AOAC instead of the recommended Lactobacilli MRS broth.